The Impact of Vitamin D Supplementation on Vitamin D Level, Urinary Calcium Excretion and Bone Density in Patients with Hypercalciuria and Vitamin D Deficiency − Preliminary Report

Abstract Aim To study the impact of vitamin D supplementation on vitamin D concentration in plasma, calcium urinary excretion and bone density in patients with urolithiasis in the course of idiopathic hypercalciuria and with a low vitamin D level. Materials and method Prospective analysis concerning 28 patients (16 boys, 12 girls) aged 6-14 years (average 10.4) in terms of urinary calcium excretion (mg/kg/day and Ca/Creatinine ratio in morning urine sample), 25OHD blood level after 3, 6, 9 and 12 months of individually recommended doses of vitamin D supplementation (400 IU or 800 IU). The doses were determined on the basis of 25 (OH) D deficiency. The children were on a normocalcemic diet. The bone mineral density of the patients was assessed before and after 12 months of vitamin D use at the aforementioned doses. Results There was no statistically significant correlation between 25 (OH) D plasma concentration and urinary calcium excretion measured on Ca /Creatinine ratio in daily urine collection and Ca/Creatinine ratio in the morning urine sample. No statistically significant change in calcium excretion was noted (measured by calciuria in daily urine collection and the calcium to creatinine ratio in the morning urine sample). A statistically significant increase in vitamin D plasma concentration was observed. Improvement in bone mineral density was not statistically significant. Conclusions Supplementation of vitamin D in the children with idiopathic hypercalciuria and urolithiasis who were examined seems to be safe. The decision to start treatment and the selection of the vitamin D dose should be considered individually. Patients with urolithiasis should be carefully monitored for calcium/phosphate metabolism parameters and the activity of the disease. Supplementation of low doses vitamin D in the children examined did not improve bone mineral density.


INTRODUCTION
A signi cant increase in the incidence of urolithiasis among the adult and pediatric population has been observed in recent years. is may be due to environmental factors, such as changing eating habits, which promote obesity and an inadequate daily intake of water.
Metabolic disorders are found in many children with urolithiasis and may promote the onset of various diseases. e most common one is idiopathic hypercalciuria (IH). It is estimated that it coexists with kidney stones in 30% -60% of the adults and 40-80% of the children a ected [1,2,3,4,5,6,7]. Idiopathic hypercalciuria is characterized by normocalcaemia and hypercalciuria and an absence of circumstances that cause increased calcium excretion (i.e. steroid therapy, use of drugs increasing calcium excretion or hyperparathyroidism). Hypercalciuria is usually de ned as daily calcium excretion above 4 mg per kilogram of body weight.
Random Ca/Creatinine ratio is a useful tool for diagnosing IH. It is a quotient of calcium and creatinine excretion in the second morning urine sample. e reference values for this ratio vary with age, latitude and range from 0.8 in infants to 0.2 in adults. Genetic predisposition to IH is suspected. Studies have shown an increased incidence of nephrolithiasis and hypercalciuria among the rst-degree relatives of patients with kidney stones [1,7,8]. In addition, diet has a large e ect on hypercalciuria in individuals with IH. Reduced intake of calcium and magnesium in the diet leads to an increase in free oxalates in the intestines, thus increased oxalate absorption and increased urinary excretion, which induce the formation of stones in the urinary tract [1,9,10]. erefore, a normocalcaemic regimen should be recommended to IH patients [9,11,12].
Vitamin D de ciency is commonly observed in the Polish population (summer months included). is may be due to changes of lifestyle and work habits (leading to lower sun exposure) and a wide use of protection against ultraviolet radiation (sunscreen). Improved methods of assessment of vitamin D supplies and the relatively low price of the test have made it possible to monitor and correct vitamin D de ciencies. Vitamin D supplementation is recommended especially in autumn and winter months. Prophylactic doses have been established for the population of our latitude [11,13].
However, vitamin D supplementation may be harmful in some patient groups. Until recently treatment was considered not safe in disorders of calcium and phosphate metabolism (urolithiasis and hypercalciuria among them). Vitamin D de ciency o en coexists with these disorders. e serum level of liver hydroxylated 25(OH)D is an indication of the body's vitamin reserves. Idiopathic hypercalciuria was proven to coexist with bone mineralization disorders (osteopenia and osteoporosis -low bone mass and impaired bone structure), hence a greater risk of bone fracture in the pediatric population [11,14,15,16]. us, the question arises whether vitamin D administration does not increase calciuria and hence promote the formation of new stones in the urinary tract among patients with IH induced urolithiasis. Another important issue is the e ect of administering strictly xed vitamin D doses on bone density in this group of patients [10,11,14].
THE AIM e aim of the study was to evaluate the impact of vitamin D supplementation on vitamin D concentration in plasma, calcium urinary excretion and bone density in patients with urolithiasis in the course of idiopathic hypercalciuria and with a low vitamin D level.

MATERIALS AND METHOD
is study included regular patients of the Pediatric, Nephrology and Allergology Department at the Military Institute of Medicine with idiopathic hypercalciuria induced urolithiasis, in which serum vitamin D concentration corresponded to an average de ciency (10-20 ng/ml) or suboptimal concentration (20-30 ng/ml) [11]. Excluded from the study were children with chronic kidney disease, urinary tract infections, urinary tract defects, systemic diseases, bone diseases (other than osteopenia), endocrine disorders and those using glucocorticosteroids.
It was planned that if control studies revealed a signi cant increase in calciuria or the formation of new deposits, then the patient would return to the previous treatment regimen (the vitamin D supplement would be withdrawn). One of the patients was treated with hydrochlorothiazide, which was discontinued at the beginning of the study. Other patients with hypercalciuria did not receive any additional treatment before and at the time of the study.
Prospective analysis included 28 patients (16 boys, 12 girls) aged 6-14 years (average 10.4). Analysis was performed of calciuria (daily calcium excretion, random Ca/Creatinine ratio), and the level of 25OHD a er 3, 6, 9 and 12 months of vitamin D supplementation. Doses were determined on the basis of 25(OH)D de ciency: 400 IU for patients with 25(OH)D 20-30ng/ml; 800 IU for those with 25(OH)D 10-20 ng/ml. Urinary calcium excretion was measured by photometric absorbance. e Ca/Creatinine ratio was calculated (quotient of calcium and creatinine excretion in the second morning urine sample).
Total concentration of 25-hydroxyvitamin D was measured with the Dia-Sorin LIAISON® analyzer, which uses chemiluminescent immunoassays (CLIA) to determine the level of 25OHD in serum and plasma.
Statistical signi cance was measured only between the initial and 12-month visit.
Bone mineral density of patients was assessed before and a er 12 months of vitamin D use at the aforementioned doses. Bone densitometry was performed with HOLOGIC model Delphi W (S/N 70608) in the Whole Body projection. Bone mineral density (BMD) in g/cm 2 was then used to calculate the T-score using the formula: Z-score = (BMD patient-BMD adjusted for age)/SD. Osteopenia is de ned as Z-score between (-2) and (-1), osteoporosis is de ned as Z score> (-2) [18].
In the rst period of observation, before the decision about vitamin D supplementation we evaluated the: − serum level of liver hydroxylated 25(OH)D, − calcium excretion in the 24h urine collection and second morning urine sample, − full body densitometry. Results were given as mean and standard deviation (Z-score). en the patients received vitamin D at a dose of 400 IU or 800 IU daily, depending on initial 25(OH) D concentration. Children with moderate vitamin D de ciency (10-20ng/ml) received 800 IU daily, children with suboptimal vitamin D concentrations (20-30 ng/ ml) received 400 IU daily.
Every 3 months, during each follow-up visit, evaluation was conducted of: − serum 25 (OH) D concentration, − calcium excretion in 24h urine collection, and Ca/ Creatinine ratio in second morning urine sample. During observation, vitamin D doses were adjusted to the seasons and concentrations of 25 (OH) D in each patient, using only a dose of 400 IU or 800 IU. Calcium excretion was not taken into consideration in the criteria of changing doses.
A er 12 months of vitamin D intake, full body bone densitometry was performed alongside the blood and urine tests.
Additionally, all the patients underwent a renal ultrasound every 3 months to assess new deposit formation. e disease activity is still studied and will be presented in the future. e results were statistically analyzed based on the so ware StatSo , Inc. (2014) STATISTICA (data analysis so ware system), version 12. Before the analysis began, the data were initially veri ed using the normality diagram of the distribution, and nally using the normality test of Kolmogorow-Smirnov and Liliefors. e distribution of data for analysis was normal, so Pearson's linear correlation coe cient was used. Statistically signi cant values were those for which p <0.05.

RESULTS
No patient in the study group required withdrawal of vitamin D supplementation. e results are shown in Figures 1, 2 and 3 and in Table I.
Urine calcium excretion, evaluated in mg/kg/day, initially amounted to 4.15 (0.17-7.44); a er 3 months, the excretion decreased to an average of 3.96 (0.79 to 10.2); a er 6 months it did not change signi cantly by an average of 3.92 (from 0.93 to 7.6); a er 9 months it rose to an average of 4.65 (from 1.83 to 7.78); whereas a er 12 months it decreased to an average of 3.57 (from 1.24 to 5.54). ere was no statistically signi cant increase in urinary calcium excretion measured before treatment and a er 12 months of treatment (p=0,61).

Ryc. 2. Wydalanie wapnia z moczem w DZM wyjściowo i po 3, 6, 9 i 12 miesiącach obserwacji.
Calcium excretion measured by Ca/Creatinine ratio was initially 0.22 (0.083 to 0.44); a er 3 months of vitamin D supplementation Ca/Creatinine ratio decreased to an average of 0.17 (from 0.041 to 0.258); a er 6 months its value did not increase and was on average 0.16 (from 0.71 to 0.29); a er 9 months an average of 0.18 was observed (from 0.063 to 0.298); a er 12 months − an average 0.18 (from 0.06 to 0.24) was registered. Urinary calcium excretion measured with Ca/Creatinine at the beginning of the study and a er 12 months of treatment did not change in a statistically signi cant way, it seemed to be even lower than initially (p=0.05).
Initially 25OHD concentration (in ng/ml) in the patients was on average 17.8 (6.8 to 31.8); a er 3 months of supplementation it increased to an average of 24.7 (from 11 to 40.2); a er 6 months it was on average 26.2 (16.1 to 54.2); a er 9 months the average was 31.14 (from 14.6 to 48.2); a er 12 months it averaged 27.59 (from 15.6 to 38.5). e increase in 25 (OH) D a er 12 months of treatment was statistically signi cant (p=0,00005) ( Figure 1, Table I).
For the evaluation of densitometry, the Whole Body Z score was used, which before treatment was on average (-1.79) [from (-1.98) to (1.46)], whereas a er one year of observation, an average of (-0.85) [from (-1.87) to (2.01)] was observed. A change in the Whole Body Z-score a er 12 months of vitamin D supplementation was not statistically signi cant (p=0.68). ere was no statistically signi cant correlation between vitamin D 25 (OH) concentration and calciuria in mg per kg body weight in daily urine collection ( Figure 1, Figure 2, Table II). No statistically signi cant correlation was found between 25 (OH) D concentration and Ca/Creatinine ratio in the daily and second morning urine sample (Figure 1, Figure 3, Table  III). e increase in bone density was not statistically signi cant (Table I, IV).

DISCUSSION
Until recently, the administration of vitamin D to patients with urinary stones was considered harmful and assumed to lead to an increase in the disease's activity [20]. Expanded interest in the value of vitamin D and its pleiotropic activity led to research into the advantageous e ects of vitamin D, especially in patients with urolithiasis coexisting with decreased bone density. In accordance with generally accepted recommendations, IH patients should have a normocalcaemic regimen [9,11,12]. e e ect of the administration of vitamin D on the activity of urolithiasis is still controversial. ere are no unequivocal studies about the in uence of vitamin D supplementation on urolithiasis severity. ere is also no clear opinion about the e ect of vitamin D supplementation on bone density in these patients.
In the literature there are only few, relatively recent studies referring to adults, and these publications present contradictory results [10,11,14].
e Department of Pediatrics, Pediatric Nephrology and Allergology at the Military Institute of Medicine takes care of the constantly increasing group of children and adolescents with urinary stones in whom serum 25 (OH) D concentration is routinely evaluated.
Observation of the common vitamin D de ciency in this group has become the basis for planning the current study with low doses of vitamin D in children with urolithiasis caused by idiopathic hypercalciuria. Vitamin D was administered under the supervision of their calcium metabolism and 25 (OH) D concentration.
is study demonstrated that 12 months of lowdose vitamin D supplementation (400 or 800 IU/day) in children with IH did not signi cantly a ect calciuria as measured by daily calcium excretion and Ca/Creatinine ratio. However, a statistically signi cant increase in vitamin D serum hepatic metabolite concentration was proven. Low doses of vitamin D did not improve bone density (Z-score) (p=0.68).
Leaf et al. [17] who carried out research on adult populations in the USA did not prove an increase in calcium excretion in patients with urolithiasis through the supplementation of high doses of ergocalciferol (50 IU per week). Johri et al. [18] found that the supplementation of vitamin D increased the excretion of calcium. However, many studies of healthy subjects treated with vitamin D supplementation did not reveal any increased risk for calciuria [19].
Zerwekh is of the an opinion that the e ect of vitamin D on the kidneys requires further research, as increased reabsorption of calcium in the renal tubules associated with increased serum vitamin D concentration may a ect decreased calcium urinary excretion [20]. us, perhaps, there is no physiological evidence for concern that vitamin D administration increases calciuria. All of these references emphasize that even low-dose supplementation of vitamin D results in a signi cant increase in serum 25 (OH) D concentration [17,18,19]. e relationship between urolithiasis and IH and decreased bone density was con rmed. However, there is no clear opinion about the e ect of vitamin D supplementation on bone density in pediatric patients. Tasca et al. believe idiopathic hypercalciuria coexists with decreased bone density and it has a strong connection with intestinal absorption, bone metabolism and calcium excretion [8]. Artemiuk et al. conducted a study on a pediatric population with urolithiasis showing a correlation between low vitamin D levels and decreased bone density in the lumbar spine [22].
A limitation of our study is the small population. Pre-quali ed patients o en did not report for screening, which unfortunately limited the number of those who completed the program. ere is also insu cient insight into vitamin D supplementation regularity, because the preparations were used by patients themselves in home conditions and only the observation of increased 25(OH)D concentration was evidence of accomplishing the recommendations.
It is possible that no improvement in bone density parameters in the presented study is associated with the low vitamin D doses or too short a follow-up period.
In subsequent studies, the use of larger doses of vitamin D should be considered. e in uence of these doses on the bone density and urolithiasis activity in children with idiopathic hypercalciuria and osteopenia should be analyzed.

CONCLUSIONS
1. e supplementation of vitamin D in the children with idiopathic hypercalciuria and urolithiasis who were examined seems to be safe. 2. e decision to start treatment and the selection of the vitamin D dose should be considered individually. 3. Patients with urolithiasis should be carefully monitored for calcium/phosphate metabolism parameters and the activity of the disease. 4. Supplementation of low doses vitamin D in the children examined did not improve bone density.